Transfusion-associated acute lung injury (TRALI), currently the leading cause of transfusion- associated fatality, is triggered by transfusion of blood products containing leukocyte antibodies and/or biological response modifiers to susceptible patients. Although many different leukocyte antibodies have been shown to induce TRALI, those specific for the leukocyte antigen HNA-3a are especially prone to cause very severe, and often fatal reactions. Unfortunately, it has not been possible to screen blood donors routinely for anti-HNA-3a because its molecular properties have for many years eluded investigators and because HNA-3a was thought to be neutrophil-specific, a cell difficult to use in serologic studies. Thus, very little is known about the prevalence of anti-HNA-3a in blood donors and the immunogenicity of this antigen is poorly understood. Even less is known about antibodies that recognize HNA-3b, the allele of HNA-3a, although they should, in theory, be as likely to cause TRALI as anti-HNA-3a. We recently showed that HNA-3a is carried on choline transporter- like protein 2 (CTL2) and that the HNA-3a/b polymorphism is almost certainly determined by an R>Q154 (R=HNA-3a, Q = HNA-3b) amino acid substitution in the first extracellular loop of the 10- membrane-spanning CTL2 protein. These findings suggest ways to develop a practical assay for detection of anti-HNA-3a and anti-3b suitable for routine donor screening. In this application, we propose to generate recombinant and synthetic CTL2 and CTL2 fragments containing R154 and Q154 and characterize their reactions against a panel of HNA-3-specific antibodies. Constructs found to be most sensitive and specific for anti-CTL2 detection will be produced in quantity, formatted into a prototype solid phase immunoassay, and used to screen 7,000 serum samples from transfused, non-transfused and parous males and females to determine the prevalence of anti-HNA-3a in these populations. The Q154 version of these constructs should provide comparable information about anti-HNA-3b. Findings made are expected to 1) define, for the first time, the prevalence of anti-HNA-3a and anti- HNA-3b in blood donor populations;2) characterize the immunogenicity of HNA-3 and HNA-3b (likelihood of an antibody being induced upon exposure to antigen through transfusion or during pregnancy) and 3) establish a basis for determining whether routine screening of blood donors for anti-HNA-3a and anti-HNA-3b is warranted. PUBLIC HEALTH RELEVANCE: Transfusion-associated acute lung injury (TRALI) is a serious complication of blood transfusion and the most common cause of transfusion-related mortality. Transfusion of antibodies specific for white blood cells (leukocytes) is a major cause of TRALI. Antibodies against a leukocyte antigen designated HNA-3a are especially prone to cause severe, often fatal TRALI but, for technical reasons, it has not been possible to test blood donors to detect them. Recent findings made in our laboratory make it likely that this obstacle can be overcome and studies to accomplish this are described in this application.))